Nucleic acid sequencing

From MIBBI

1. Nucleic acid preparation

1.1. Molecule type

1.2. Extraction method¹

1.3. Amplification method²

1.4. Cleanup³

2. Library construction

2.1. Library size⁴

2.2. Library reads sequenced⁵

2.3. Library vector⁶

2.4. Library screening strategy⁷

3. PCR amplification

3.1. Target gene⁸

3.2. Target subfragment⁹

3.3. PCR primers¹⁰

3.4. Multiplex identifiers¹¹

3.5. PCR conditions

3.5.1. Denaturation¹²

3.5.2. Annealing¹³

3.5.3. Elongation¹⁴

3.5.4. Final elongation¹⁵

4. Sequencing

4.1. Sequencing method¹⁶

4.2. Sequence quality check¹⁷

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Footnotes

¹ Link to a literature reference, electronic resource or a standard operating procedure (SOP) through a DOI, PMID or URL.

² Link to a literature reference, electronic resource or a standard operating procedure (SOP) through a DOI, PMID or URL.

³ Link to a literature reference, electronic resource or a standard operating procedure (SOP) through a DOI, PMID or URL.

⁴ The total number of clones in the library prepared for the project.

⁵ The total number of clones sequenced.

⁶ The cloning vector types used to construct the library.

⁷ Specific enrichment or screening methods applied before and/or after creating clone libraries.

⁸ Targeted gene or locus name for marker gene studies.

⁹ Name of subfragment of a gene or locus. It is important, for example, to identify special regions on marker genes like V6 on 16S rRNA.

¹⁰ PCR primers, barcodes and adaptors that were used to amplify the sequence of the targeted gene or locus. If multiple forward or reverse primers are present in a single PCR reaction, describe all the primers used. Primer sequence should be reported in uppercase, except barcode or adaptor sequence, which should be in lowercase.

¹¹ Molecular barcodes, called Multiplex Identifiers (MIDs), that are used to specifically tag samples in a sequencing run. Their sequence should be reported in uppercase.

¹² Provide time and temperature for this step.

¹³ Provide time and temperature for this step.

¹⁴ Provide time and temperature for this step.

¹⁵ Provide time and temperature for this step.

¹⁶ The type of sequencing used.

¹⁷ Indicate if the sequence has been called automatically or has undergone a manual editing procedure (e.g., by inspecting the raw data or chromatograms). Applies only to sequences that are not submitted to SRA,ENA or DRA.

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Sources

MIGS

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Notes